Oncogenes

The c-myc gene belongs to the Myc gene family and functions as a gene-specific transcription factor through its protein product, c-Myc, for a wide range of human cancers. The c-Myc protein regulates almost 20% of all cellular genes and is also involved in cell cycle regulation, apoptosis, metabolism, cellular differentiation, and cell adhesion[49]_{. As a result, the}

aberrant over expression of c-myc is associated with a variety of malignant tumors including those of breast, colon, cervix, and myeloid leukemia[50]_{. In particular, c-Myc has been}

identified as one of the main activating factors for the human telomerase reverse transcriptase (hTERT) catalytic domain of the telomerase enzyme[51]_{. The nuclear hypersensitivity}

element III1 (NHE III1) upstream of the P1 promoter of c-myc is a G-rich strand containing a

27-base-pair sequence (Pu27, Table 1.1), which has the propensity to adopt a G-quadruplex structure. The presence of a quadruplex within this promoter region was initially proposed based on chemical probe studies, gel mobility measurements, and fluorescence resonance energy transfer (FRET) spectroscopy[52]_{. In later studies, the topological structures of several c-}

myc quadruplex sequences were determined by circular dichroism (CD), NMR, and mutational experiments [16a, 53]_{. The G-rich region of c-myc contains more than four}

consecutive G-strands, resulting in the formation of a dynamic mixture of four parallel G- quadruplex loop isomers in the native Pu27 region. Furthermore, two different sequences

derived from the Pu27 region have been analyzed by NMR[20d]_{, revealed that both myc-2345}

(Table 1.1) and myc-1245 (Table 1.1) fold into intramolecular propeller-type G-quadruplexes in K+ _{solution. In this case, the core of three G-tetrads is formed by four G-stretches oriented in}

the same direction, with all the guanines in an anti glycosidic conformation and the three loops adopting double-chain-reversal structures, very similar to the crystalline state of telomeric G-quadruplex conformation in K+_{. Similar structures have also been found in the}

Pu24I (Table 1.1) and myc22-G14T/G23T (Table 1.1) sequences [54]_{. A interesting structural}

feature of Pu24I revealed by NMR studies show that a guanine base (G24) at the 3 end plugs back into the G-tetrad core by participating in G-tetrad formation and displacing another guanine (G10) of a continuous guanine tract in a loop. This configuration is maintained by a stable diagonal loop, which contains a G•(A-G) triad stacking on and capping the G-tetrad core. These new folding features result from the presence of five guanine tracts in the sequence that are different from the four guanine tracts in the c-myc sequences studied previously.

bcl-2 is a proto-oncogene, and its oncogenic property arises from decreasing the rate of cell death[55]_{. Its protein product, Bcl-2, is a mitochondrial membrane protein, which is present}

in delicate balance with related proteins and is involved in the control of programmed cell death, functioning as an apoptosis inhibitor [56]_{. Over-expression of bcl-2 has been found in a}

wide range of human cancers, including B-cell and T-cell lymphomas, breast, cervical, prostate, and colorectal. In addition, it also functions in chemotherapy-induced apoptosis[55]_,

which indicates its potential role in drug resistance. The human bcl-2 gene contains a GC-rich region upstream of the P1 promoter, which is critical for the regulation of bcl-2 gene expression. It can form a mixture of three distinct intramolecular G-quadruplexes (5 G4, MidG4, and 3 G4) resulting from the six runs of guanines, including one run of five guanines, two runs of four guanines each, and three runs of three guanines each (Table 1.1). With more than four consecutive G-tracts in the sequence, the G-quadruplex in bcl-2 has the ability to

form either three or six different loop isomers [57]_{. The central G-quadruplex (MidG4, Table}

1.1), which is the most stable of the major species formed in the bcl-2 promoter region, is likely to form a mixed parallel/antiparallel structure consisting of three tetrads connected by loops and to give rise to three possible loop isomers. An NMR study of the shorter and mutated bcl-2 quadruplex bcl2MidG4Pu23-G15T/G16T (Table 1.1) has shown that one of the topologies for this mixed parallel/antiparallel intramolecular quadruplex has two lateral loops and one propeller loop, similar to one of the Tel22 telomeric quadruplex topologies [20c, 58]_{. The}

G-rich strand located in the bcl-2 P1 promoter plays a significant role in the regulation of bcl- 2 transcription [59]_{. Although the effects of G-quadruplex ligands on bcl-2 expression remain}

to be deciphered, some studies have shown that some G-quadruplex ligands can induce apoptosis [60]_{. In particular, the ligand 12459 has been found to induce apoptosis characterized}

by dysfunction of Bcl-2 [61]_{. These findings suggest the possible role of G-quadruplex formation}

in the bcl-2 promoter during apoptosis.

Recently, two G-rich sequences (c-kit native and c-kit21, Table 1.1) in the promoter region of the human c-kit gene have been identified, and biophysical studies have shown that these sequences can form G-quadruplexes [20e, 62]_{. In the c-kit native sequence, 87 base pairs}

upstream of the transcription start site of the human c-kit gene, a single G-quadruplex structure forms in K+ _{solution. An NMR study has shown that the c-kit87up (Table 1.1)}

sequence forms a new intramolecular G-quadruplex[63]_{. Most strikingly, an isolated guanine}

(G10) is involved in G-tetrad core formation, despite the presence of four three-guanine tracts. There are four distinctive loops, including two single-residue and double-chain-reversal loops (A5, C9), a two-residue loop (C11, T12), and a five-residue stem-loop (A16, G17, G18, A19, G20). In view of the importance of predicting G-quadruplex topologies from sequence information, these new folds in which G residues in non-G-tract regions can participate in structural core formation are particularly worthy of attention. In the case of the c-kit21

sequence, a variety of quadruplex conformations have been identified. This sequence needs to be mutated in order to form a single quadruplex species, probably with a parallel fold.